This rat C-peptide-2 differs from the active human C-peptide by several amino acids, however conserved Glu at positions 3, 11, and 27 is essential for peptide activity. In rat medullary thick ascending limb, C-peptide stimulates Na+,K+-ATPase activity within a physiological concentration range. This effect is due to an increase in Na+,K+-ATPase turnover rate that is most likely mediated by protein kinase C-f phosphorylation of the Na+,K+-ATPase f-subunit.